Localization of G6PD and HPRT to different arms of the X chromosome of the North American marsupial (Didelphis virginiana) by in situ hybridization and deletion mapping: evolutionary significance

We have mapped HPRT and G6PD loci on the X chromosome in the American opossum, Didelphis virginiana, by in situ hybridization to cells derived from two females by using genomic opossum DNA as probes. The localizations (G6PD to Xp13 and HPRT to Xq21), indicating that the two genes are separated by the centromere, were confirmed by results of hybridization to X chromosomes with deletions that include the HPRT locus and opossum-mouse cell hybrids containing the relevant fragment of the opossum X chromosome.     

Molecular studies of marsupial X chromosomes reveal limited sequence homology of mammalian X-linked genes

To explore the extent to which the X chromosome has been conserved during mammalian evolution, we compared six loci that are X-linked in the human genome with the corresponding genes of the North American marsupial, the Virginia opossum (Didelphis virginiana). Our analysis shows that in the opossum genome there are sequences highly homologous to those of human cDNAs for housekeeping genes, glucose-6-phosphoribosyltransferase (HPRT), phosphoglycerate kinase A (PGK1), and alpha-galactosidase A (GLA). However, ornithine transcarbamylase and blood clotting Factor IX--tissue-specific genes that are X-linked in eutherians mammals--have no highly conserved homologs in the marsupial genome. By cloning opossum G6PD and HPRT, we found that these genes are X-linked in the opossum and that homologous sequences are limited to coding regions. As all genomic fragments hybridizing with the human GLA probe show dosage effects, it is likely that the opossum counterpart is X-linked. Finally, the pattern of hybridization suggests that the autosomal pseudogenes of HPRT and PGK1 in the opossum have remained highly homologous to the human X-linked genes.     

A study of X chromosome regulation during oogenesis in the mouse

Mature oocytes of mammals, in contrast to somatic cells, have two active X chromosomes. This situation might arise through either of two possible mechanisms. The germ line might be differentiated from somatic cells prior to X inactivation. Alternatively, an X chromosome in germ cells would be reactivated after prior inactivation. This paper presents data compatible with reactivation of the X in germ cells. X-linked enzymes were compared in oocytes of XX and XO fetal mice. The activity of G6PD is similar in the two classes of cells at early meiotic stages, but an $\text{XX}\text{XO}$ ratio of 2:1 is approached at later times; this suggests reactivation of the G6PD locus. For HPRT, a 2:1 ratio is observed at all meiotic stages. HPRT shows a large increase in enzyme activity during early meiosis, while G6PD does not. Synthesis of this enzyme at early meiotic stages probably accounts for differences between these data and those obtained for G6PD, and places the time of X reactivation at the entry to meiosis.     

Frequent derepression of G6PD and HPRT on the marsupial inactive X chromosome associated with cell proliferation in vitro

X chromosome dosage compensation in Marsupials is like that in eutherian mammals except that the paternal X chromosome is always inactive, and silence of this chromosome is not well maintained. We previously showed that the unstable inactivation of the paternal G6PD allele is associated with the lack of DNA methylation in the 5' CpG cluster. Even though this CpG island is unmethylated, the paternal allele (marked by an enzyme variant) is at least partially and often severely repressed in most tissues of the opossum, so that factors other than methylation must inactivate the locus. Here we report that when cell cultures are established from these tissues, the silent G6PD locus is depressed. Although often complete, the extent of derepression differs among tissues and within different cell types in the same tissue, and is not accompanied by obvious changes in the pattern of chromosome replication. Studies of the HPRT locus in these cells show that the paternal HPRT allele also derepresses in cultured cells. These observations suggest that without DNA methylation to maintain the silence of the locus, tissue or cell-specific factors act to repress the silent locus, but are unable to maintain inactivity through cell division, or are lost as cells proliferate in culture.     

Frequency of reactivation and variability in expression of X-linked enzyme loci.

A mouse-human cell hybrid clone retaining an inactive human X chromosome was treated with 5-azacytidine. Following treatment, expression of the X-linked enzyme markers, hypoxanthine-guanine phosphoribosyltransferase (HPRT), glucose-6-phosphate dehydrogenase (G6PD), phosphoglycerate kinase (PGK), and alpha-galactosidase A (GLA) was examined. Results presented here show that 45 of the 62 clones positive for human HPRT expressed human GLA, while only four of 68 clones negative for human HPRT expressed human GLA. These results strongly suggest that there is coordinate reactivation of GLA and HPRT. Reactivated expression of G6PD was studied in detail. The studies show that 5-azacytidine can induce heritable changes in the inactive human X chromosome resulting in the expression of G6PD activity at a level lower than that from an active human X chromosome.     

Regional localization of the genes for human HEXB. PGK, GALA. HPRT, G6PD by somatic cell hybridization

Twenty independent man-mouse (Cl1D,LA/TK-, HPRT-) and man-hamster (CH,HPRT-) hybrids using female human cells with balanced reciprocal translocation XX,t(X;5)(q21;q11) were analyzed for human genes localized on chromosome 5 (HEXB), on chromosome X (PGK, GALA, HPRT, G6PD) and for the different chromosomes in relation with the balanced reciprocal translocation (chr.5, chr.5q-, chr.Xq+, chr.X). The different results obtained indicate that the genes for human markers HEXB, PGK are on Xq+, and that the genes for human markers GALA, G6PD are on 5q-. These data implicate finally the following localizations: HEXB on 5q11 leads to 5qter; PGK on Xq21 leads to Xpter; GALA, HPRT, G6PD on Xq21 leads to Xqter.     

Chromosome 9 of Ellobius lutescens is the X chromosome

Ellobius lutescens carries an apparently identical karyotype (2n = 17) in both sexes. On the basis of indirect evidence the unpaired chromosome 9 has been considered to represent the X chromosome of this species. We have obtained data to substantiate this view by four different techniques. After fusion of HPRT^– RAG cells with E. lutescens fibroblasts we demonstrated that the enzymes HPRT and G6PD are localized on the presumptive X chromosome. By analysis of pachytene figures after silver staining we showed by electron microscopy that the single chromosome exhibits the typical features of an X chromosome in male meiosis. Hybridization of (GATA)₄ and (GACA)₄ oligonucleotide probes to E. lutescens DNA revealed several distinct bands in the high molecular weight range some of which appeared to be specific for the individual but not for the sex of the animal. Hybridization in situ of the (GATA)₄ probe on metaphase spreads of E. lutescens did not highlight any particular chromosome segment but showed a significant deficit of these sequences in chromosome 9. These observations are discussed with respect to their bearing on X chromosome determination. Finally it is concluded that E. lutescens should be an ideal tool for testing candidate genes assumed to be involved in primary sex determination.     

Segregation of rat chromosomes in somatic cell hybrids between rat cells and HT 1080 human fibrosarcoma cells

Summary We produced somatic cell hybrids between HT 1080-6TG human fibrosarcoma cells and either rat white blood cells (WBC) or cells directly derived from rat spleen. Karyologic and isozyme analyses of hybrid cells indicated that they preferentially lose rat chromosomes. Hypoxanthine-aminopterine thymidine-selected hybrid clones expressing rat hypoxanthine phosphoribosyltransferase (HPRT), glucose-6-phosphate dehydrogenase (G6PD), and phosphoglycerate kinase (PGK) and containing the rat X chromosome were counterselected in a medium containing 30 g/ml of 6-thioguanine. Concordant loss of the rat X chromosome and of the expression of rat HPRT and G6PD was observed in the hybrid clones.     

雌性畸胎瘤细胞离体分化时X染色体的行为

用雌性畸胎瘤LT细胞(具有两个X染色体)为材料,在离体条件下诱导分化。通过对X连锁的HPRT和G6PD等酶的定量分析,并与Pcc3/A/1畸胎瘤细胞(XO型)对比。结果表明,HPRT与G6PD酶比活性在分化后的LT细胞中,以及在已分化的胚胎体重新种植并传代后的细胞中,均与Pcc3/A/1(XO型)细胞相似,比未分化的细胞降低了一半左右。这些结果可认为,在雌性畸胎瘤细胞离体分化过程中,发生了X染色体的生化分化。     

Old and new genetics help ordering loci at the telomere of the human X-chromosome long arm

Summary A Sardinian pedigree described in 1964 for having been found to segregate at the X-linked loci for the Xg^a antigen, G6PD deficiency, Protan and Deutan color blindness, with an instance of recombination between the last two loci, was re-examined with respect to four common X-linked DNA polymorphisms detected by molecular probes homologous to critical subregions of the human X chromosome. Two branches of this pedigree-including the one with the Protan-Deutan recombinant-were found to segregate also for the common BamHI polymorphism identified with the cDNA probe pHPT-2 of the HPRT gene (Xq26). The analysis of the chromosome haplotypes in the male offspring of the phase known penta-heterozygous mother suggests that the probable order of the relevant loci is HPRT, Deutan, G6PD, Protan, Xq telomere. Though we are fully aware of the risks of generalizing the significance of observations made on a single exceptional pedigree, we believe that this report outlines the potential of families of the type described as reasearch tools to resolve the linear order of tightly X-linked loci and to investigate the biology of genetic recombination in humans.     

Chinese hamster x American mink somatic cell hybrids: characterization of a clone panel and assignment of the mink genes for malate dehydrogenase,NADP-1 and malate dehydrogenase,NAD-1

Summary Chinese hamster x American mink somatic cell hybrids were obtained and examined for chromosome content and expression of mink malate dehydrogenase, NADP (MOD-1; EC 1.1.1.40), malate dehydrogenase, NAD (MOR-1; EC 1.1.1.37), glucose-6-phosphate dehydrogenase (G6PD; EC 1.1.1.49) and hypoxanthine phosphoribosyltransferase (HPRT; EC 2.4.2.8). All the hybrid clones examined were found to segregate mink chromosomes. A clone panel containing 25 clones was set up. The possibilities and limitations of this panel for mink gene mapping are analysed. Using this panel, it is feasible to rapidly map genes located on chromosomes 1–13 and to provisionally assign genes located on chromosome 14 and the X. Based on the data obtained, the genes for MOD-1 and MOR-1 were firmly assigned to mink chromosomes 1 and 11, respectively, and the genes for G6PD and HPRT were provisionally assigned to the X.     

Creation of a clone panel of fox x Chinese hamster somatic cell hybrids and chromosome mapping of genes for LDHA, LDHB, GPI, ESD, G6PD, HPRT, alpha-GALA in the silver fox

A clone panel of fox-hamster somatic cell hybrids which can be used for fox gene mapping was set up. Analysis of patterns of chromosome-enzyme segregation made it possible to assign gene GPI to chromosome 1, LDHA to chromosome 11, LDHB to chromosome 8, ESD to chromosome 6 and G6PD, HPRT, alpha-GALA to chromosome X.     

Subchromosomal localization and order of GLA, PGK1, HPRT, and G6PD loci on the X chromosome of the American mink (Mustela vison)

Segregation of the X-linked mink markers alpha-galactosidase (GLA), phosphoglycerate kinase-1 (PGK1), hypoxanthine phosphoribosyltransferase (HPRT), and glucose-6-phosphate dehydrogenase (G6PD) was analyzed in hybrids of gamma-irradiated mink fibroblasts and Chinese hamster cells and in hybrids of nonirradiated mink fibroblasts and mouse hepatoma cells. Based on this analysis, the order of the four genes is GLA-PGK1-HPRT-G6PD on the mink X chromosome. Cytogenetic analysis of five mink x Chinese hamster hybrid clones containing mink GLA, PGK1, and HPRT, but lacking G6PD, tentatively localized mink G6PD to Xq15.22----qter and also confirmed the gene order as GLA-PGK1-HPRT-G6PD-qter. Comparison of this order with its counterpart in man and the mouse, as well as an analysis of the G-band patterns of their X chromosomes, demonstrated putative similarities between mink and man and differences in the mouse. These differences may be due to a different rate of X-chromosomal rearrangement in mammalian evolution.     

Marsupial--mouse cell hybrids containing fragments of the marsupial X chromosome.

Hybrids were obtained from fusions of HPRT-deficient mouse fibroblasts and marsupial lymphocytes. These hybrids retained no identifiable marsupial chromosomes, but all expressed the marsupial form of HPRT. Half the clones also expressed marsupial PGK-A, and half of these also marsupial G6PD; no other marsupial allozyme markers were detected. Since G6PD is known to be sex linked in these species, we conclude that Hpt and Pgk-A are also located on the X chromosome and the markers lie in the order Hpt-Pgk-A-Gpd.     

Polymorphism at the G6pd and 6Pgd loci in Drosophila melanogaster. IV. Genetic factors modifying enzyme activity

Different homozygous lines of similar genotype with respect to G6pd and 6Pgd were shown to have different enzyme activities for G6PD and 6PGD. Crosses between high and low lines suggested that there were modifying genes present on the autosomes, while others were probably located on the X chromosome. Allelic variation within each electrophoretic class of G6pd and 6Pgd might, however, also have contributed to this variation. An experiment on adaptation to sodium octanoate demonstrated that in adapted flies selection for lower enzyme activity had occurred, which provided further evidence for the existence of genetic differences in activity. Furthermore, a strong positive correlation between the activities of G6PD and 6PGD was found for each genotype. Since no correlation was found between MDH and the two enzymes G6PD and 6PGD, it could be concluded that this correlation was probably rather specific for G6PD and 6PGD. Interaction between genotypes with respect to activity was also found. It was shown that the variation at 6Pgd influenced the activity of G6PD within a genotype. The data are discussed in relation to fitness differences presented in foregoing articles.     

Human gene mapping using an X/autosome translocation.

Human fibroblasts containing a translocation between the X chromosome and chromosome 15 were fused with the 6-thioguanine-resistant mouse cell line, IR. Resulting hybrids, selected in HAT medium, retained the X/15 chromosome. Hybrids which were counterselected in 6-thioguanine lost this chromosome. The X-linked markers glucose-6-phosphate dehydrogenase (G6PD), phosphoglycerate kinase (PGK), and hypoxanthine phosphoribosyl transferase (HPRT), and the non-X-linked markers pyruvate kinase (PKM2) mannose phosphate isomerase (MPI), N-acetyl hexosaminidase A (HEXA) and beta2-microglubulin (beta2-m) all segregated in concordance with the X/15 translocation chromosome. The latter markers have been assigned to chromosome 15. Selection against the X/15 chromosome was done using antihuman beta2-m serum. Electrophoretic and immunochemical analyses of the N-acetyl hexosaminidases A and B in these hybrids were performed.     

Allelic expression in intergeneric fox hybrids (Alopex lagopus × Vulpes vulpes). III. Regulation of the expression of the parental alleles at the Gpd locus linked to the X chromosome

The electrophoretic pattern of glucose-6-phosphate dehydrogenase (G6PD) was studied in 60 intergeneric fox hybrids (Alopex lagopus × Vulpes vulpes), 33 females and 27 males. It is shown that the structural gene for G6PD, designated Gpd, is located on the X chromosome in both Arctic and silver foxes. Analysis of G6PD patterns in the erythrocytes of hybrid females demonstrated that the phenotypic expression of parental alleles at the Gpd locus varied considerably: from 1:1 to the hemizygous manifestation of an allele of either the Artic or the silver fox. The expression of the parental allels at this locus is different in the various tissues of single female hybrids. It is suggested that the variable quantitative expression of the alleles at the Gpd locus in hybrid females is related to the presence of two cell populations having in an active state either the X chromosome of the Arctic fox or that of the silver fox. It is also proposed that the size of the two cell populations is largely affected by the different relationships between cells having different activated X-chromosomes among initiator (stem) cells from which various definitive organs and tissues develop. The number of initiator cells for erythroid tissue has been calculated to be five or six.     

Regional mapping of the human No. 1 and X chromosome in interspecific cell hybrids using an X/1 translocation.

Fibroblasts from a carrier of an X/1 translocation, 46,XY,t(X;1)(q28;q31), were fused with Chinese hamster cells. The resulting hybrids were analyzed for human No. 1 and X-chromosome markers. The data indicate that the loci for PGM1, PGD, PPH, and GuK1 are situated either in the long arm proximal to a break point in band 1q31 or in the short arm. The loci for Pep-C, FH, and GuK1 are located distal to the break point. HPRT and G6PD are probably situated distal to a break point in band q28 of the X chromosome; alpha-Gal A is situated proximal to the break point, either on the long or short arm of the chromosome.